Our long-term goal is to elucidate the molecular mechanisms that control axonal guidance in the developing vertebrate central nervous system (CMS). Commissural axons project along a circumferential trajectory toward the floor plate (FP), an intermediate target at the ventral midline (VM) of the spinal cord. The roof plate (RP)-associated chemorepellent, BMP? and the FP-derived chemoattractants, Netrin-1 and Shh guide commissural axons to the VM. Cell adhesion molecules (e.g., NrCAM) appear to mediate contact dependent interactions between commissural axons and FP cells that facilitate midline crossing. Many decussated commissural axons turn rostrally and initially project alongside the FP, but never re-cross the VM. FP contact is thought to alter the responsiveness of commissural axons so that they can adopt new post-crossing trajectories. Rodent commissural axons lose their attraction to netrin-1 and gain responsiveness to the midline repellents, Slit2 and Sema3B/3F after contacting a FP, in vitro, and mice lacking Slits or their Robo receptors exhibit midline guidance defects. Decussated commissural axons apparently turn in the anterior direction by responding to opposed gradients of the FP-derived chemoattractant, Wnt4, and Shh, acting as a midline repellent. Most decussated commissural axons travel along a diagonal path away from the VM, project into an intermediate/dorsal region of the spinal cord and then execute a second rostral turn along a boundary of ephrin-B expression (ILC trajectory). Surprisingly, some commissural axons can elaborate ILC-like projections even in the absence of FP contact. The basic helix-loop-helix proteins Mathl and Ngn1/Ngn2 define separate neuronal progenitors that differentiate into genetically distinct, d!1 and d!2 commissural interneurons, respectively. Mathl, Ngn1 and Ngn2 enhancer elements direct reporter gene expression to d!1 and d!2 commissural neurons/axons in transgenic mice and chick embryos, and decussated d!1 and d!2 axons extend along ILC-like trajectories. These observations suggest that the concerted actions of long-range guidance systems (Robo-Slit, Npn-Sema, Wnt-Fz, Shh signaling) and contact-dependent guidance signals (NrCAM, PSA-NCAM, L1, Eph/ephrins) regulate the pathfinding of post-crossing d!1 and d!2 axons, even in the absence of FP contact. To test this hypothesis, the pathfinding of dM and d!2 commissural axons will be examined in mouse lacking key guidance molecules and after blockade of long-range and/or short-range guidance systems in chick embryos. Spinal cord injury severs axon tracts and compromises neuronal function. The molecular logic of axonal guidance must be understood in order to repair damaged spinal circuits. The proposed studies should provide a basis for devising strategies aimed at directing the re-growth of regenerating nerve fibers.